‘Fuzzy’ and ‘Tandem’ Complexes: NMR-based Molecular Understanding of Interactions of Short Linear Motifs with their Binding Modules

Protein-protein interactions (PPIs) between short linear motifs (SLiMs) and their cognate binding domains play important roles in biological regulation. Determining molecular factors contributing to the affinity of such complexes (typically intermediate-to-low) is necessary for understanding their cellular function and for efficient therapeutic targeting. Using solution NMR we have characterized two systems involving ubiquitous motif-binding domains. First, we provide first structural proof that the SH3 domain of cortactin, a nucleation promoting factor responsible for actin polymerization and a biomarker for invasive cancers, binds a proline-rich segment of WASp-interacting protein (WIP). Structure determination of the WIP/cortactin complex reveals a dual binding mode, including key interactions with a hydrophobic surface adjacent to canonical binding grooves. The multiple binding poses, or ‘fuzzy’ complex, is typical of regulatory PPIs, and offers an intriguing comparison to similar complexes solved in our previous studies. Second, we study the interaction between a double WW-domain of tumor suppressor oxidoreductase (WWOX) and the receptor tyrosine kinase ErbB4 containing multiple PPxY binding motifs. WWOX is intriguing because its WW1 is partially unfolded, and its WW2 lacks a Trp residue necessary for binding. A combination of NMR and isothermal calorimetry provides insight into the binding of single- and double-PPxY motifs to this tandem WW domain, including the orientation of the two proteins, the effects of binding competition and avidity, and the contribution of inter-motif linking segments on formation of the complex. These structural insights have directed the design of motifs with increased affinity, underlining their importance in potential pharmaceutical applications.